Post-Reductionist Protein Folding: Determining the In-cell Folding Energy Landsca

内质网（ER）是细胞的折叠工厂，每秒能够产生数千种分泌蛋白。这些蛋白质的错误折叠导致从囊性纤维化到肝硬化等疾病。虽然全长蛋白可以在体外使用无数生物物理技术进行研究，从而使生物物理学家能够详细表征其折叠能量景观，但在更生理相关的内质网环境中研究蛋白质折叠存在令人生畏的技术障碍。利用最近开发的强大的单分子荧光技术，研究含有荧光标记氨基酸的新生多肽链，我们将确定新生多肽链在内质网中延长时的构象演变，以及与内质网中的伴侣和修饰酶的相互作用如何调节构象空间。目标是获得关于生长的新生链折叠景观的详细信息，与试管实验中可用的数据相媲美。内质网驻留蛋白和新生链之间的共和翻译后相互作用可能使折叠能量景观平滑，对错误折叠和易聚集的中间体产生偏倚。这种折叠格局的重塑对于具有高接触顺序的蛋白质尤其重要，即在链的远端部分之间有许多接触。我们将重点关注一个重要的分泌蛋白家族，抑制性蛇形蛋白，它折叠成具有高接触顺序的天然结构。有趣的是，蛇的原生状态在能量上是亚稳态的，可以转变成不同的结构。蛇形蛋白通过调节包括凝血和炎症在内的关键生理过程中的蛋白酶发挥重要的生物学作用。一些与肝硬化和肺气肿（统称为“蛇形蛋白病”）等疾病相关的蛇形蛋白突变导致内质网中的蛇形蛋白聚集。通过研究serpin在内质网中的折叠，并将其与体外折叠进行比较，我们将确定与内质网蛋白的相互作用以及疾病相关突变如何改变折叠景观。这项研究的结果将提供前所未有的洞察蛇形蛋白是如何错误折叠的，以及它们的能量景观在体外和体内是如何不同的。这项工作还将为研究内质网中蛋白质折叠的科学家提供一个容易获得的实验工具箱，并为治疗丝状病变和其他内质网折叠疾病的潜在治疗策略提供一个平台。

The endoplasmic reticulum (ER) is a folding factory for the cell, able to churn out thousands of secretory proteins per second. Misfolding of a number of these proteins results in diseases ranging from cystic fibrosis to liver cirrhosis. While full-length proteins can be studied in vitro using a myriad of biophysical techniques, thus allowing biophysicists to characterize in detail their folding energy landscapes, studying protein folding in the more physiologically relevant ER environment presents daunting technical obstacles. Using recently developed, powerful single molecule fluorescence techniques on nascent polypeptide chains that contain fluorescently-labeled amino acids, we will determine the conformational evolution as a nascent polypeptide chain elongates in the ER and how conformational space is modulated by interactions with the chaperones and modifying enzymes resident in the ER. The goal is to obtain detailed information on the folding landscape of the growing nascent chain, rivaling the data available in test-tube experiments. Co- and post-translational interactions between the ER-resident proteins and nascent chains likely smooth the folding energy landscape, biasing against misfolded and aggregation-prone intermediates. This remodeling of the folding landscape is particularly important for proteins with high contact order, i.e. many contacts between distal parts of the chain. We will focus on an important family of secreted proteins, the inhibitory serpins, which fold into native structures with high contact order. Intriguingly serpin native states are energetically metastable, poised to change to a different structure. Serpins play crucial biological roles by regulating proteases involved in key physiological processes including blood coagulation and inflammation. A number of serpin mutations, associated with diseases such as liver cirrhosis and emphysema (together called 'serpinopathies'), lead to serpin aggregation in the ER. By studying serpin folding in the ER and comparing it to in vitro folding, we will determine how the folding landscape is altered by interactions with ER-resident proteins and by disease-associated mutations. The results of this research will provide unprecedented insight into how serpins misfold and how their energy landscapes differ in vitro and in vivo. This work will also provide a readily accessible experimental toolbox for scientists studying protein folding in the ER as well as a platform for potential therapeutic strategies to treat serpinopathies and other ER folding diseases.